Highly efficient gas permeable devices and methods for culturing cells

ABSTRACT

This invention relates to methods and devices that improve cell culture efficiency. They include the use of gas permeable culture compartments that reduce the use of space while maintaining uniform culture conditions, and are more suitable for automated liquid handling. They include the integration of gas permeable materials into the traditional multiple shelf format to resolve the problem of non-uniform culture conditions. They include culture devices that use surfaces comprised of gas permeable, plasma charged silicone and can integrate traditional attachment surfaces, such as those comprised of traditional tissue culture treated polystyrene. They include culture devices that integrate gas permeable, liquid permeable membranes. A variety of benefits accrue, including more optimal culture conditions during scale up and more efficient use of inventory space, incubator space, and disposal space. Furthermore, labor and contamination risk are reduced.

RELATED APPLICATION

The present application claims priority to U.S. application Ser. No.17/164,977 filed Feb. 2, 2021, which is a continuation of U.S.application Ser. No. 15/643,621 filed Jul. 7, 2017, issued as U.S. Pat.No. 11,377,635, which is a continuation of U.S. application Ser. No.14/321,933 filed Jul. 2, 2014, issued as U.S. Pat. No. 9,732,317, whichis a continuation of U.S. application Ser. No. 11/952,848 filed Dec. 7,2007, issued as 8,809,044 which claims the benefit of U.S. ProvisionalApplication No. 60/873,347 filed Dec. 7, 2006, both of which areincorporated herein in their entirety by reference.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, and all of the patents andco-pending applications naming John Wilson as an inventor, are herebyexpressly incorporated herein by reference. More generally, documents orreferences are cited in this text, and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference.

GOVERNMENT INTERESTS

This invention was made in part with U.S. Government support underNational Institutes of Health Small Business Innovative Research GrantDK0659865 “Islet culture, shipping, and infusion device”. The U.S.Government may have certain rights in this invention.

1. TECHNICAL FIELD

This invention relates to methods and devices that improve cell cultureefficiency. They include the use of gas permeable culture compartmentsthat reduce the use of space while maintaining uniform cultureconditions, and are more suitable for automated liquid handling. Theyinclude the integration of gas permeable materials into the traditionalmultiple shelf format to resolve the problem of non-uniform cultureconditions. They include culture devices that use surfaces comprised ofgas permeable, plasma charged silicone and can integrate traditionalattachment surfaces, such as those comprised of traditional tissueculture treated polystyrene. They include culture devices that integrategas permeable, liquid permeable membranes.

2. DISCUSSION OF LIMITATIONS OF CONVENTIONAL TECHNOLOGIES DESCRIBED INRELATED ART

The culture of cells is a central element of biotechnology. Tissueculture flasks are devices commonly used for cell culture because theyallow adherent and non-adherent cell types to be cultured in them, aredisposable, and can function in a static mode without need for equipmentto perfuse medium. Traditional flasks have one culture compartment.Their design requires a gas-liquid interface to be present within thedevice for gas exchange of the culture.

Culture medium must reside at a very low height so that oxygen deliveryto the cells is not compromised. The height of medium that isrecommended for tissue culture flasks is between 2 mm and 3 mm. However,the body of the flask must be large enough to hold gas and allow accessto the medium, typically by use of a pipette. Thus, flasks have a largedevice volume relative to the amount of medium they contain. Forexample, the body of a typical T-175 flask has a footprint approximately23 cm long by 11 cm wide, is about 3.7 cm tall, and therefore occupiesabout 936 cm³ of space. However, it typically operates with about 50 mlof medium. Thus, the medium present in the flask body (50 ml), relativeto the space occupied by the flask body (936 cm³) demonstrates that onlyabout 5% of the flask's volume is occupied by medium. Furthermore,dividing the volume of space occupied by the body of the flask (936 cm³)by the surface area for cells to reside upon (175 cm²) shows that thevolume of space occupied by the flask is over 5 times the surface areait provides for cells to reside upon. Flasks are manufactured withvarious amounts of surface area for cells to reside upon, typicallyranging from 25 cm² to 225 cm² in area, and therefore only have a smallculture capacity. As more and more flasks are used during culture scaleup, the overall amount of space they occupy relative to the small mediumvolume and limited culture surface area they provide creates aninherently inefficient use of space that burdens the culture processwith excess cost related to shipping, sterilization, storage, incubatorspace, and disposal. This problem is compounded by the substantiallyincreased labor and contamination risk.

Multi-shelved flasks, such as the NUNC Cell Factory (U.S. Pat. No.5,310,676) and CORNING® CELLSTACK® (U.S. Pat. No. 6,569,675), attempt toaddress inefficient flask scale up by stacking shelves in the verticaldirection to create multiple culture compartments within one flask. Thiscreates more surface area within one device and therefore allows morecells to reside in the multi-shelved flask than the traditional flask.In this manner, one multi-shelved flask can replace numerous traditionalflasks. The multi-shelved flask can be configured so that medium can beaccessed through a common collection point, precluding the need forpipette access to each culture compartment. That allows the distancebetween each shelf of the multi-shelved flask to be reduced relative tothe height of the traditional flask. For example, the space betweenshelves of the NUNC Cell Factory is about 1.4 cm, as opposed to the 3.7cm distance between the bottom and top of a typical T-175 flask creatingsome improvement in the use of storage, shipping, sterilization,culture, and disposal space. A vent in the multi-shelved flask allowsgas exchange with the ambient atmosphere in order to adjust pH, provideoxygen, and to help maintain temperature control. However, gas at anygiven location within the multi-shelved flask resides at a differentdistance from the vent location. Since the distance between gas at thefarthest point and gas at the closest point to the vent increases as thenumber of culture compartments within the multi-shelved flask isincreased during scale up, gradients in CO2 and O2 concentrations candevelop throughout the gas within the multi-shelved flask. Therefore,the multi-shelved flask design has an inherent potential for non-uniformculture conditions to exist throughout the device and the problem iscompounded during scale up.

There are a number of static cell culture devices that perform gastransfer by making the lower wall of the device gas permeable. Gasdiffuses through the gas permeable lower wall in response toconcentration gradients that develop between the culture medium and theambient gas. This approach eliminates the gas-liquid interface as thesole source of gas exchange. Since the surface that cells reside upon isgas permeable, more uniform culture conditions can exist throughout theculture than the multi-shelved flask. Bags are static gas permeabledevices that integrate a single culture compartment. To scale a cultureup, the bag must elongate in the horizontal direction to create moresurface area for cells to reside upon. Thus, they quickly becomeunwieldy and outsize cell culture incubators during scale up. Bags arecommercially available from OriGen Biomedical Group (ORIGEN PERMALIFEBags), Baxter (LIFECELL® X-FOLD related to U.S. Pat. Nos. 4,829,002,4,937,194, 5,935,847, 6,297,046 B1), Medtronic

(SI-CULTURE, U.S. Pat. No. 5,686,304), Biovectra (VECTRACELL), andAmerican Fluoroseal (VUELIFE Culture Bag System, covered by U.S. Pat.Nos. 4,847,462 and 4,945,203). Gas permeable cartridges are devices thatoperate in the same manner as bags, except they have rigid sidewalls.Commercially available gas permeable cartridges include CLINICELL®Culture Cassettes provided by Laboratories MABIO-INTERNATIONAL® andOPTICELL® gas permeable cartridges (U.S. Pat. Nos. 6,455,310 and6,410,309) provided by BioChrystal Ltd. As with bags, in order toprovide more surface area for cells to reside upon, these devices mustelongate in the horizontal direction. In U.S. Pat. No. 6,821,772, theinventor of OPTICELL® has proposed multiple gas permeable compartments.Unfortunately, the proposal merely increases the number of culturecompartments in the horizontal direction. Thus, regardless of the numberof culture compartments, increasing the culture capacity of thesedevices requires that they be made larger in the horizontal direction.None of these gas permeable devices are capable of scaling in thevertical direction.

In an attempt to utilize space more efficiently, U.S. Pat. No. 6,673,595describes the scale up of OPTICELL® gas permeable cartridges by stackingindividual, physically distinct, cartridges in the vertical directionand handling each individual cartridge with a very complex automatedsystem. This scale up approach deviates markedly from the simplicityafforded by the traditional multi-shelved flask.

U.S. Pat. No. 6,759,245 described a multilayered gas permeable culturedevice that separates oxygen delivery from medium delivery by use of agas permeable, liquid impermeable membrane. This invention is based onthe discovery that if the flows of liquid medium and oxygenated fluidare separated by a gas permeable, liquid impermeable membrane, and thecells are grown attached to the liquid side of the membrane, the devicecan be used to culture cells with the transport of oxygen through themembrane without regard for the flow rate of liquid medium passingthrough the device. The advantage being that the flow rate of liquidmedium is no longer dependent on the need to carry oxygen to the cells.However, although the flow of medium is substantially lowered, as it isonly needed to carry substrates such as glucose, it precludes theability to culture suspension cells since they will be washed from thedevice during use. In this approach, cells must be attached to acollagen matrix. Another disadvantage is the need to perfuse the gasspace and/or the liquid space. This requires pumps, fluid lines, and agreatly elevated level of complexity relative to traditional multipleshelf flasks. Thus, this approach has not been commercialized.

Gas permeable devices that make more efficient use of space aredescribed in co-pending U.S. patent application Ser. No. 10/961,814(Wilson et al.). Among the gas permeable devices described in Wilson etal. '814 are those that allow culture scale up in the vertical directionwhile retaining the simplicity of the traditional multi-shelved flask.For example, Wilson et al. '814 describe the vertical scale up of gaspermeable devices comprised of shelves stacked one above the other forcells to reside upon. Gas transfer occurs through the walls of thedevice. Unlike the scale up of traditional gas permeable devices,increasing culture size can be achieved by increasing the size of thedevice in the vertical direction as opposed to the horizontal direction.Since there is no need for a gas-liquid interface, this allows optimalspace efficiency during vertical scale up of a culture. A more compactdevice is attained relative to the multi-shelved flask. Attributes notpossible in the traditional multi-shelved flask are present. Forexample, the device can be inverted to allow adherent cells to becultured on the upper and lower surfaces of the stacked shelves tofurther optimize space efficiency. The invention described hereinexpands upon the gas permeable advantages described in co-pending Wilsonet al. '814 to create new geometry that provides a superior alternativeto the traditional multiple shelf flask.

It is an object of the present invention to provide improved cellculture devices and methods that minimize the potential for non-uniformculture conditions to exist throughout the device, allow space efficientculture scale up of adherent or suspension cells, are easy to use, canfunction without need to perfuse medium or gas, and allow the user tomake effective use of the upper, lower, or sidewall surfaces of eachculture compartment. Still further objects and advantages will becomeapparent from consideration of the ensuing description and drawings.

SUMMARY OF THE INVENTION

The present invention overcomes many of the disadvantages of existingstatic cell culture devices by integrating at least two gas permeableculture compartments that, at least in part, maintain a gas spacebetween them in order to allow gas to contact the gas permeable area ofthe culture compartments. This allows each culture compartment toexchange gas directly with the gas space adjacent to the culturecompartment, minimizing the potential for non-uniform cultureconditions. Selected surfaces of the culture compartments can be madegas permeable to provide gas exchange on the surface opposite cellsand/or adjacent to cells. Surfaces inside the culture compartments canbe comprised of various materials to provide optimal surfaces for cellsto reside upon. Surface area inside the culture compartments can beincreased if desired, such as may be the case when adherent cells orcells that thrive in a three dimensional matrix are cultured. It is alsopossible for cells to reside directly upon the gas permeable material ofthe culture compartments. Scaling the device can be accomplished byadding culture compartments such that, at least in part, a gas spaceexists between each culture compartment in order to allow gas to contactthe gas permeable area of the culture compartments. Access to theculture compartments can occur by way of a common manifold, commonmanifolds, or by discrete access to each compartment. With thisconfiguration, it is possible to scale cultures in a simple format thatis easy to use, makes efficient use of space, and minimizes thepotential for non-uniform culture conditions to occur. Various featurescan be included, and configurations can be structured, to provideadditional benefits including the ability for the device to be operatedin more than one position, allow the culture of adherent cells, allowthe culture of suspension cells, allow co-culture, prevent cells fromexiting their respective culture compartments during routine handling,minimizing feeding frequency, replicate traditional flask protocols,allow the surface area for cells to reside upon to be increased ordecreased during culture, allow the ratio of medium volume to thesurface area for cells to reside upon to be increased or decreasedduring culture, and/or to allow the cells to reside on or in proximityof alternative materials.

In one aspect of the present invention, each culture compartmentincludes a first wall and an opposing second wall, the first wall and/orthe second wall being comprised of gas permeable material, and a gasspace is present between at least a portion of each culture compartment.

In another aspect of the present invention, each culture compartmentincludes several walls, including but not limited to a first wall and anopposing second wall, a third wall and an opposing fourth wall, and afifth wall, the first wall and/or second wall and/or third wall and/orfourth wall and/or fifth wall being comprised of gas permeable material,and a gas space is adjacent to at least the gas permeable portion ofeach culture compartment.

In another aspect of the present invention, the culture compartments areconnected in parallel by one manifold. The manifold can be configured toprevent gas from displacing medium held within the culture compartments,and/or can be configured to retain cells in the culture compartmentsduring handling, and/or can be configured to retain medium and gas inthe culture compartments.

In another aspect of the present invention, the culture compartments areconnected in parallel by more than one manifold.

In another aspect of the present invention, the height of the culturecompartments can change.

In another aspect of the present invention, a culture compartmentsupport resides between culture compartments to maintain the culturecompartments in a substantially horizontal position and/or allow gas tocontact the gas permeable surfaces of the culture compartments.

In another aspect of the present invention, walls of the culturecompartments include projections that make contact with at least one ofits neighboring culture compartments in order to maintain the culturecompartments in a substantially horizontal position and allow gas tocontact the gas permeable surfaces of the culture compartments.

In another aspect of the present invention, structure is provided toprevent walls of the culture compartments from making contact withneighboring walls of the culture compartment.

In another aspect of the present invention, the culture compartments areconnected in sense.

In another aspect of the present invention, direct access to each of theculture compartments is possible.

In another aspect of the present invention, contact between ambient gasand the gas space of the gas permeable multi-shelf device can beselectively terminated, restricted, or unrestricted.

In another aspect of the present invention, a method of expanding cellsfrom one culture compartment to multiple culture compartments ispossible.

In another aspect of the present invention, when the gas permeablemulti-shelf culture device is oriented such that cells are residing onthe lower most culture surfaces of the culture compartments, at least aportion of one culture compartment does not have a culture compartmentdirectly above it in order to facilitate microscopic evaluation.

In another aspect of the present invention, when the gas permeablemulti-shelf culture device is oriented such that cells are residing onthe lower most culture surfaces of the culture compartments, the gasspace between the lowest culture compartment and the culture compartmentresiding above it allows light to be present above the lowest culturecompartment to facilitate inverted microscopic evaluation of the lowestculture compartment.

In another aspect of the present invention, a method of co-culturingcells is possible by seeding cells to a culture surface andrepositioning the device to allow another inoculum of cells to gravitateto a different culture surface.

In another aspect of the present invention, a method of culturing cellson a particular surface, at a particular oxygen tension, and aparticular medium height, and/or at a particular medium volume tosurface area ratio is available by merely rotating the device toreposition the cells from surface to surface. It is also possible toculture at least five different cell lines, each residing on a differentwall of the culture compartment.

In another aspect of the present invention, culture compartments arefabricated as an integral unit to minimize the number of seals.

In another aspect of the present invention, the gas permeablemulti-shelf device can be configured to retain the features ofcommercially available, traditional multiple shelf flasks whileresolving the problems of non-uniform culture conditions.

In another aspect of the present invention, the use of gas permeable,liquid permeable materials are disclosed for use in a gas permeable cellculture device that includes a culture compartment support and asterility barrier between the gas space and the ambient gas.

In another aspect of the present invention, structuring gas permeabledevices with plasma charged silicone for the purpose of minimizingmigration to other surfaces is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B illustrate an embodiment of a gas permeablemulti-shelf device that is configured for gas exchange to occur directlythrough the walls of the culture compartments.

The culture compartments are connected in parallel with a manifold toform an integral unit, including the presence of a gas space between theculture compartments. An access port allows fluid to move into and outof the gas permeable multi-shelf device.

FIG. 2 shows a cross-sectional view of one embodiment of a gas permeablemulti-shelf device that is configured to allow gas transfer through afirst wall of the culture compartments in order to allow gas exchangevia the surface of the medium that resides the furthest distance fromcells.

FIG. 3 shows a cross-sectional view of one embodiment of a gas permeablemulti-shelf device that is configured to allow gas transfer through asecond wall of the culture compartments in order to allow gas exchangein proximity of the cells.

FIG. 4 shows a cross-sectional view of one embodiment of a gas permeablemulti-shelf device that is configured to allow gas transfer through afirst wall and through a second wall of the culture compartments inorder to increase the surface area available for gas exchange.

FIG. 5A, FIG. 5B, and FIG. 5C show a perspective view of a gas permeablemulti-shelf device that is configured with a first wall and an opposingsecond wall, a third wall and an opposing fourth wall, and a fifth wall.By selectively fabricating each wall with a predetermined length, width,surface area, and material composition, and each culture surface of apredetermined material and surface area, the user can select from avariety of culture protocols. Thus, by merely altering the orientationof the gas permeable multi-shelf device, the user can expose cells,various types of culture surfaces, culture surface areas, oxygentension, and medium volume to surface area ratios. For example, when thegas permeable multi-shelf device is oriented as shown in FIG. 5B, mediumcan reside at a greater height than when the gas permeable multi-shelfdevice is oriented as shown in FIG. 5A When the gas permeablemulti-shelf device is oriented as shown in FIG. 5C, medium can reside ata height that exceeds what is possible when the device is oriented asshown in FIG. 5A or FIG. 5B.

FIG. 6 shows a cross-sectional view of a gas permeable multi-shelfdevice that is configured with a gas trap to prevent gas from displacingmedium from within the culture compartments.

FIG. 7A and FIG. 7B show a cross-sectional view of one embodiment of agas permeable multi-shelf device that is configured to prevent cellsfrom exiting the culture compartments during routine handling. FIG. 7C,FIG. 7D, and FIG. 7E show a cross-sectional view of one embodiment of agas permeable multi-shelf device that is configured to allow medium andgas to reside in the culture compartments at predetermined volumes.

FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8C-1 show a cross-sectional view ofone embodiment of a gas permeable multi-shelf device that is configuredwith two manifolds. Culture compartments are connected in parallelbetween manifolds, forming an integral unit that includes a gas spaceadjacent to each culture compartment. FIG. 8D and FIG. 8E show how thevolume of medium and/or the gas permeable surface area to culturecompartment volume ratio can be altered before or during the cultureprocess.

FIG. 9A and FIG. 9B illustrate the use of a culture compartment supportfor the purpose of allowing gas to contact the gas permeable material ofthe culture compartments and/or for the purpose of maintaining theculture compartments in a substantially horizontal position in order toallow cells to distribute uniformly within the culture compartments.

FIG. 10 illustrates the use of a culture compartment support in the formof external projections emanating from the walls of the culturecompartments for the purpose of allowing gas to contact the gaspermeable material of the culture compartments and/or for the purpose ofmaintaining the culture compartments in a substantially horizontalposition in order to allow cells to distribute uniformly within theculture compartments.

FIG. 11 illustrates the use of an internal spacer situated within theculture compartments for the purpose of preventing walls, and/or culturesurfaces from contacting each other.

FIG. 12 shows a cross-sectional view of one embodiment of a gaspermeable multi-shelf device wherein culture compartments are connectedin series with inlet and outlet ports. The culture compartments form anintegral unit and include a gas space adjacent to each culturecompartment.

FIG. 13 shows a cross-sectional view of one embodiment of a gaspermeable multi-shelf device that is configured with individual culturecompartment and with a gas space residing adjacent to each culturecompartment. It also shows how ambient gas access to the gas space canbe selectively controlled.

FIG. 14 shows another embodiment of a gas permeable multi-shelf devicethat is configured to control ambient gas access to the gas space.

FIG. 15 shows another embodiment of a gas permeable multi-shelf devicethat is configured to control ambient gas access to the gas space.

FIG. 16A and FIG. 16B show a method of using a gas permeable multi-shelfdevice to expand cells from one culture compartment to multiple culturecompartments.

FIG. 17A shows a view of an integral culture compartment molded ofsilicone that minimizes the number of joints for leakage. FIG. 17B showsan integral group of culture compartments fabricated as a single piece.FIG. 17C shows an exploded view of a gas permeable multi-shelf devicethat includes integral culture compartment, overmolded flanges, and twomanifolds, and culture compartment supports.

FIG. 18A shows a traditional multiple shelf flask manufacturersrecommended approach to solving the problem of non-uniform cultureconditions. FIG. 18B shows a magnified view of that approach. FIG. 18C,FIG. 18D, and FIG. 18E show embodiments of gas permeable multi-shelfdevices configured to resolve the problem of non-uniform cultureconditions while integrating the features of commercially available,traditional multiple shelf flasks. In one embodiment, gas transferoccurs directly through the walls of the device, allowing direct gasexchange between the gas above each culture compartment and that of theambient environment. In another embodiment, a gas space resides withinthe device, allowing gas exchange of the gas within the device throughthe walls of the gas space. The upper wall of the gas space can beadapted to allow gas transfer independent of a gas-liquid interfaceand/or the lower wall of the gas space can be adapted to allow gastransfer to the culture by way of a gas-liquid interface. In anotherembodiment, the gas space is in communication with ambient gas by way ofa gas permeable device wall, and a gas permeable upper wall of the gasspace acts as the lower wall of the culture compartment, allowing gastransfer to occur to the culture independent of the gas-liquidinterface.

FIG. 19 shows a histogram of data collected from islet culture, a verychallenging type of culture due to the high oxygen demand of islets. Thesuperior results demonstrate the unique capacity of the gas permeablemulti-shelf device to save space and provide uniform culture conditionswhen including beneficial geometry into its culture compartmentsupports.

FIG. 20 shows a cross-sectional view of a test fixture used todemonstrate the usefulness of plasma charging materials comprised ofsilicone prior to exposing the gas permeable multi-shelf device totypical sterilization processes such as gamma irradiation in order tominimize the migration of silicone to other surfaces.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1A and FIG. 1B are useful for illustrating some of the basicattributes of the invention. In these illustrations, gas permeablemulti-shelved device I integrates two gas permeable culture compartments20 separated in part by gas space 50. Although two culture compartmentsare shown to allow a description of some of the basic gas permeablemulti-shelf device attributes, any additional number can be provided.FIG. 1A shows a perspective view with a sidewall removed to expose theinside of culture compartments 20. FIG. 1B shows a cross-sectional viewof gas permeable multi-shelf device 1. Culture compartments 20 arecomprised of a first wall 110 and an opposing second wall 120. Firstwall 110, second wall 120, or both first wall 110 and second wall 120can be comprised of gas permeable material. First wall 110 and secondwall 120 can be secured together without need of a sidewall, such as isthe case with traditional cell culture bags. However, a sidewall ispreferred to allow a uniform distance between the first and second wallsof the culture compartments to exist throughout the culturecompartments. Furthermore, when selected walls and sidewalls arecomprised of gas permeable material, the gas permeable multi-shelfculture device can be oriented such that it functions in a variety ofpositions including first wall down, second wall down, or any of thesidewalls down. This allows cells to reside on, in proximity of, any ofthe walls. When the length, height, and width of the culturecompartments differ in dimension, the gas permeable multi-shelf culturedevice can provide a unique medium height above cells and/or a uniquemedium to surface area ratio by altering its position during inoculationand/or culture. Depending on orientation during inoculation, cells cangravitate to any surfaces within the culture compartments. For example,in the position of FIG. 1B, cells gravitate toward second wall 120. Thesurface that cells contact can simply be the inner surface of theculture compartment wall.

However, the desired material composition and the geometry of materialthat cells reside upon may not be that presented by the inner surface ofthe culture compartment walls. In this event, any component, insert,matrix, or the like that provides the desired material and geometry canbe structured into the gas permeable multi-shelf device. Thus, culturesurface 130 can simply be the inner surface of a given culturecompartment wall, or can be any component, insert, matrix, and the likethat resides within the culture compartment. Although not limiting thescope of the invention, but merely for convenience, throughout thisapplication culture surface 130 is depicted on the inner surface of thedevice walls.

As shown in FIG. 1B, manifold 60 creates a fluid pathway between culturecompartments 20. Access port 70 allows fluid and cells to be added andremoved. In this illustration, access port 70 includes a neck and cap 30covers access port 70 in the manner of a traditional flask. However, theaccess port(s) can be any configuration(s), and can be located in anylocation(s), that meet the objective of moving fluid into and out of thegas permeable multi-shelved device. Those skilled in the art of cellculture device design will recognize that there are many ways to achievethat objective, including many closed system configurations which mayinclude the use of septums, quick disconnect fittings, or tubingconfigured for sterile splicing.

Gas space 50 need not be an enclosed aspect of the device. It need nothave forced gas flow, or be adapted for forced gas flow, in order forthe device to function. In the simplest and preferred form, it is justambient gas in contact with any or all of the gas permeable portions ofthe device. However, one or more walls can surround it.

In a simple method of operation, medium and cells are delivered into thegas permeable multi-shelf device, and gas permeable multi-shelf deviceis placed into a standard cell culture incubator, oriented such thatcells gravitate to the desired surface. In a more complex mode ofoperation, additional inoculations can be undertaken to allow cells togravitate to additional surfaces. For example, by periodicallyrepositioning the device during inoculation, cells can reside on allculture surfaces.

Each culture surface 130 can be any suitable material, and any shape,that is useful for culturing cells and may be or may not be integral tothe walls of the culture compartments. For example, the culture surfacecould simply be the inner surface of the wall that comprises the culturecompartment, and may be tissue culture treated or not. It could bematerial that is laminated to the wall of the culture compartment suchas described in U.S. Pat. No. 5,935,847. It could be a material that isphysically separate from the wall of the culture compartment, such as aseparate part fabricated of polystyrene that resides upon the wall, andmay or may not be affixed to the wall, such as a fibronectin or acollagen matrix insert. There is no restriction on the use of anyculture surface that is known to those skilled in the art of cell andtissue culture.

FIG. 2 illustrates how uniform culture conditions can be establishedwith gas exchange across gas permeable first wall 110 of culturecompartments 20. Oxygen flux arrows 100 show how, as a result of anoxygen concentration gradient between medium 80 and gas space 50, oxygenis delivered across gas permeable first walls 110 to cells 90 residingon culture surface 130 by way of medium 80. Depending on how much offirst wall 110 is gas permeable, up to virtually the entire uppersurface of the medium can be simultaneously exposed to the ambient gassurrounding the device, unlike the traditional multiple shelf flask inwhich ambient gas enters the device at a single gas permeablelocation(s) that resides at a different distance from every mediumlocation within the device. As described herein, in some embodiments,the gas permeable multi-shelf device is structured with the ability forgas and medium to reside in the culture compartments. Thus, gas canreside on both sides of the gas permeable first wall 110 duringoperation. In general, gas moves into and out of the culturecompartments due to the partial pressure differential between the gasspace and the fluid within the culture compartment.

FIG. 3 illustrates gas exchange across gas permeable second walls 120 ofthe culture compartments. Oxygen flux arrows 100 show how, as a resultof a concentration gradient between medium 80 and gas space 50, oxygenis delivered through gas permeable second walls 120 to cells 90 residingin culture compartments 20. In this manner, cells in each cellcompartment are closer to the ambient gas than is possible in thetraditional multi-shelved flask. Care should be taken to ensure that thematerials that comprise culture surface 130 do not impede gas transferbeyond what is needed to oxygenate desired number of cells and maintainproper pH. For example, if gas permeable second walls 120 areconstructed of a material with high gas transmission, such as dimethylsilicone, and a less gas permeable culture surface such as polystyreneresides upon the silicone, gas transfer to the cells will be impeded. Ingeneral, the material most resistant to gas transmission that residesbetween the cells and the ambient gas source will be rate limiting.Thus, to optimize the function of the gas permeable multi-shelf device,the design should contemplate the gas transmission of the materials usedto construct gas permeable walls, the gas transmission of any additionalculture surfaces that are used, and the oxygen demand of the culture.The gas permeable multi-shelf device allows many options for providingacceptable gas exchange and acceptable materials for cells to resideupon. However, if a material is desired for the culture surface thatwill restrict gas exchange of the culture, it is possible to enhance gasexchange by way of the opposing wall, and/or the sidewalls of theculture compartment.

FIG. 4 illustrates gas exchange across gas permeable second walls 120and across gas permeable first walls 110 of the culture compartments.Oxygen flux arrows 100 show how, as a result of a concentration gradientbetween medium 80 and gas space 50, oxygen is delivered to culturecompartments 20 by way of gas permeable second walls 120 and gaspermeable first walls 110. In this manner, a high level of gas transferis available to each culture compartment.

Although FIG. 2 , FIG. 3 , and FIG. 4 show gas transfer through specificwalls, any wall of the gas permeable multi-shelf device can be gaspermeable. A variety of advantages become available because oxygen canbe delivered to cells directly through the surface they reside upon,and/or through the sidewalls of the culture compartments, and/or throughthe manifold wall(s).

Wilson et al. '814 describe the advantages that can be obtained byincreasing the height of medium that can reside in a gas permeableculture compartment. Medium height in the gas permeable multi-shelfdevice can increase far beyond the 2 mm to 3 mm limits of traditionalflasks, thereby minimizing the frequency of medium exchange, reducinglabor, and reducing contamination risk. Thus, when gas transfer occursacross a gas permeable wall of a culture compartment, it may bebeneficial to structure the culture compartment so that the distancebetween the gas permeable wall and the opposing wall allows mediumheight to increase. The optimum distance will depend upon the metabolicdemand of the culture and the desired frequency of medium exchange.

FIG. 5A, FIG. 5B, and FIG. 5C illustrate an example of how a gaspermeable multi-shelf device can be positioned to allow many cultureoptions. For example, when first wall 110, and/or second wall 120,and/or third wall 122, and/or fourth wall 124, and/or fifth wall 126 arecomprised of material of differing gas permeability, cells can come toreside upon at a different oxygen tension by orienting gas permeablemulti-shelf device 2 to a given position. However, the gas permeablematerial need not differ to allow robust operating protocols. Theculture surface, integral or non-integral, in proximity of first wall110, and/or second wall 120, and/or third wall 122, and/or fourth wall124, and/or fifth wall 126 can differ in material and/or surface area.

When the dimensions of first wall 110, third wall 122, and fifth wall126 differ, orienting gas permeable multi-shelf device 2 in any positionalso allows the maximum height of medium to change at any point beforeor during culture. Altering the shape of the cell compartments cancreate even more options. For example, an octagonal shape allowsadditional surfaces for cells to reside upon, as the device isreoriented.

To advance the objective of establishing uniform culture conditions inthe gas permeable multi-shelf device, the design should include theobjective of placing an approximately equal number of cells within eachculture compartment, and facilitating an approximately uniformdistribution of those cells throughout each culture compartment. Makingthe geometry of each culture compartment virtually identical,structuring the opposing walls of each culture compartment to beapproximately parallel, and allowing the culture compartments to residein a horizontal position so that cells can gravitate uniformly upon theculture surface can help achieve that objective. Then, when cells are ina uniform suspension during inoculation, and the culture surface is ofuniform geometry, the inoculum will reside at a uniform volume above theculture surface of each culture compartment and cells will settle in auniform distribution upon the culture surface of each culturecompartment. In the case where culture surfaces are not flat, such aswhen corrugated surfaces are present, configuring the culturecompartments to have an equal volume of space above each unit of culturesurface area can assist uniform cell distribution during inoculation.For example, if the culture surface was corrugated and the opposing wallwas also corrugated, the volume of space between the corrugated opposingwall and the culture surface would remain constant along the length ofthe culture compartment. Regardless of culture surface geometry,configuring the culture compartments so that an approximately equalvolume of inoculum is present at any given section within the culturecompartment can help achieve uniform cell distribution.

Preferably, when a manifold is used to deliver medium to the culturecompartments, the manifold should be structured to allow inoculum todistribute evenly into each culture compartment and to minimize thenumber of cells that settle within the manifold. Making the volume ofmanifold no larger than needed to allow medium to quickly and easilyfill the culture compartments is beneficial, since cells residing in thevolume of medium retained in manifold will settle to the bottom ofmanifold and not be at the identical culture conditions as cellsresiding in the culture compartments. Although manifold volume should beminimized during inoculation to prevent cells from gravitating toundesired areas, it can be useful to allow excess volume of medium toreside in the manifold to reduce device height, since that medium cancontribute to the ratio of medium volume to surface area within eachculture compartment. Stated differently, medium volume in the manifoldcan make substrates available to cells residing in the cellcompartments.

FIG. 6 shows a cross-sectional view of gas permeable multi-shelf culturedevice 3 configured to locate gas that may become present within thedevice into an area where it does not disrupt the establishment ofuniform culture conditions. Manifold 60 includes gas trap 61. At least aportion of gas trap 61 is elevated higher than the uppermost culturecompartment 20. In this illustration, access port 70 is covered byseptum 72. Excess gas in the device rises to gas trap 61, therebyavoiding displacement of medium from any of the culture compartments.

In some applications, it may be desirable to alter the shape or volumeof the manifold during use. Structuring the manifold to change shape orvolume should be done in a manner that does not allow contamination,such as may be achieved by flexible walls or the use of gaskets oro-rings. For example, it may be desirable to deliver cells to culturecompartments by way of a common manifold and prevent cells from movingfrom one culture compartment to another, or from a culture compartmentinto the manifold. When the device is to be handled in a manner that mayorient the culture compartments in a position that would inadvertentlyallow cells to exit the culture compartment. Blocking the opening, oropenings, of the culture compartments can prevent that. As anotherexample, it may be helpful to alter the volume of medium residing in themanifold at some point during use, as may be the case when cells haveattached within the culture compartments and more medium volume isuseful for minimizing the feeding frequency. In this case, the manifoldcan be structured to increase in volume. In other applications, notfilling the culture compartment entirely with medium may beadvantageous, as may be the case when the desired culture surface areato medium volume ratio dictates that medium should reside at a heightthat is lower than the height of the culture compartment. FIG. 7A andFIG. 7B show an illustration of how these objectives may beaccomplished. In FIG. 7A, manifold wall 62 of gas permeable multi-shelfdevice 4 is in a first position that allows cells and medium to beintroduced into culture compartments 20 by way of manifold 60 via accessports 70. FIG. 7B shows manifold wall in a second position in whichmanifold 60 has collapsed to block the opening of culture compartments20, preventing cells or medium from exiting culture compartments 20.Culture compartments 20 can be partially filled with medium so thatmedium and gas reside in the culture compartments 20, and manifold wall62 can be moved into the position of FIG. 7B to prevent loss of mediuminto manifold 60. However, this embodiment also allows gas permeablemulti-shelf device 4 to be entirely filled with medium without need ofmoving manifold wall 62.

FIG. 7C, FIG. 7D, and FIG. 7E show an example of how to partially fillthe culture compartments with medium. FIG. 7C shows how gas permeablemulti-shelf device 4 can be positioned with manifold 60 oriented belowculture compartments 20 and residing in a first open position with apredetermined volume of medium 80 residing in it. The predeterminedvolume of medium is less than the combined volume of the culturecompartments. FIG. 7D shows manifold 60 compressed to drive medium 80from manifold 60 and into culture compartments 20. FIG. 7E shows gaspermeable multi-shelf device 4 oriented horizontally, such that medium80 and gas reside in each culture compartment. When the manifold isclosed, the internal volume of the gas permeable multi-shelf device isreduced, increasing pressure. The pressure increase is related to theratio of gas to liquid in the culture compartments, the compliance ofthe culture compartment walls, and the volume of the manifold to thevolume of the device. The pressure can eventually be reduced dependingon which surfaces of the culture compartments are gas permeable.

However, sterile venting of the manifold as it is reduced in volume willrelieve pressure more quickly. Those skilled in the art will recognizethat there are many ways to structure the manifold to meet theseobjectives including the use of flexible walls, rigid walls structuredwith an o-ring in a radial seal arrangement, and other approachesincluding methods described in Wilson U.S. Pat. No. 7,229,820.

Movement of manifold wall 62 can also be useful when medium is reducedin temperature during use. For example, the culture of islets is ofteninitiated at 37C and then reduced to 22C. When the gas permeablemulti-shelf device is a closed body and filled with medium, medium willcontract as temperature drops. Many gas permeable materials are highlyflexible. Thus, the walls of the device can move to maintain contactwith the medium when medium contracts. When the walls move, and cellsare uniformly distributed on the walls, cells can be displaced from auniform position to uncontrolled density and thus the viability of theculture can be compromised. Therefore, the ability to alter the volumeof the manifold to accommodate a reduction in medium volume can preventthe displacement of the cells from their uniform position.

If desired, feet 135 can elevate the gas permeable multi-shelf device.Feet 135 allow gas to access the underside of the device and/or preventscratches to the second wall 120. Feet 135 can be present in anyembodiment and the upper wall of the device can be adapted to allow onedevice to reside above the other in an interlocking manner.

Connecting the culture compartments in parallel with more than onemanifold can allow gas to be more easily displaced by liquid enteringthe device. For example, when one manifold is used, gas is displaced inthe opposite direction of medium entering the manifold. As the height ofculture compartments is reduced in a gas permeable multi-shelved devicewith one manifold, tilting the gas permeable multi-shelved device canbecome necessary to expedite the displacement of gas. Creating anadditional manifold can allow the gas to displace in a direction otherthan that at which medium is entering the device and can reduce oreliminate the need for tilting, thereby simplifying automated fluidhandling. In test fixture evaluations intended to determine if culturecompartments can be primed without need of tilting the device, primingwithout tilting was achieved when the volume of medium in the manifoldswas about 7.0% of the total volume in the test fixture. FIG. 8A, FIG.8B, and FIG. 8C show one embodiment that utilizes two manifolds. FIG. 8Ashows gas permeable multi-shelf device 5 with a wall removed to exposeculture compartments 20. Gas space 50 resides between culturecompartments 20. In this illustration, gas space 50 is an openingthrough the entire body of gas permeable multi-shelf device 5. FIG. 8Bshows gas permeable multi-shelf device 5 with a section removed toexpose culture compartments 20, first wall 110, and manifold 60. FIG. 8Cand FIG. 8C-1 show cross-sectional view 8C-8C of FIG. 8A, exposingculture compartments 20, gas space 50, and manifold 60. In thisembodiment, as liquid enters access port 70 and primes manifold 60 andculture compartments 20, gas is displaced via another manifold 60, onthe distal end of culture compartments 20, and a secondary access port70.

The height of the culture compartments can vary to allow a wider varietyof protocols. For example, it may be beneficial if the volume of trypsinused is minimized, or medium height is increased. FIG. 8D and FIG. 8Eillustrate one configuration for how that can be accomplished by simplymaking the device of flexible material that is pleated so that theculture compartment(s) 20 can rise or collapse in height. For examplethe housing and/or material bounding gas spaces can be flexible. In thismanner, the gas permeable multi-shelf device can be expanded toaccommodate more, or less, volume in each culture compartment, which maybe desired to allow a reduction in feeding frequency, reduced use oftrypsin and/or PBS, and/or a change in the gas permeable surface area toculture compartment volume. In this depiction, manifold walls 62 arepleated, but the device can be adapted to allow a change in culturecompartment height by a variety other means, including those describedin U.S. Pat. No. 7,229,820. Skilled artisans will recognize a variety ofways to allow this attribute.

A factor in optimal performance of the gas permeable multi-shelf deviceis the orientation of the culture compartments during use. During use,the gas permeable multi-shelf device should preferably be in asubstantially horizontal position for uniform cell distribution onto thecell culture surface. The culture compartment support may be as simpleas culture compartment support 40, shown in FIG. 5A In this case,culture compartment support 40 merely provides simple structural supportto prevent culture compartments from collapsing upon each other.However, depending on the stiffness of the materials that comprise thewalls of the culture compartments, it may be advantageous to form a moreelaborate culture compartment support. For example, some important cellculture applications are best conducted in very controlled geometry thatis directed at the deposit of cells in very uniform distribution, suchas the culture of islets, hepatocytes, and multipotent adult progenitorcells. For example, islets will aggregate when in contact at highsurface density, and multipotent adult progenitor cells candifferentiate if they are too close to one another. Hepatocytes andislets also have a need for a high rate of gas transmission to retainhealth. Thus, the most robust culture compartment support will allow auniform distribution by maintaining the wall that cells are gravitatingto in a substantially horizontal position, and not overly restrict gastransmission. To obtain these benefits, the culture compartment supportwill make contact with the culture compartment walls. The number ofcontact points, distance between contact points, and amount of surfacearea of the gas permeable material in direct contact with the culturecompartment support are among the design factors to consider. Example 1and Example 2 provide additional guidance.

Although it can be permanently affixed to the gas permeable multi-shelfdevice, the culture compartment support does not need to be. This may bedesirable when a user has a need to convert the device for a morecontrolled application, or to reduce manufacturing cost. FIG. 9A andFIG. 9B show an embodiment in which the culture compartment support isreusable, and the body of the gas permeable multi-shelved device isdisposable. In FIG. 9A, culture compartment support 42 is shown detachedfrom gas permeable multi-shelf device 6. In FIG. 9B, culture compartmentsupport 42 has been placed in contact with gas permeable multi-shelfdevice 6.

Projections 131 emanate from culture compartment support 42. The heightand distance between projections 131 should be designed with theobjective of making enough contact with the culture compartments of thegas permeable multi-shelved device to hold the cell culture compartmentsin a substantially horizontal state such that a uniform cell deposit canbe achieved during inoculation. However, contact with the gas permeablesurfaces diminishes gas transfer capacity. Therefore, a balance betweenthe desire for a horizontal state and the degree of desired gas transfermust be considered. Depending on the type of cells being cultured, therecan be more than one optimum design. Gas access openings 132 can bepresent when more access to ambient conditions is desired. In theabsence of gas access openings 132, gas will move between the surfacethat projections 131 emanate from, such as surface(s) 133, and theculture compartment resistance to gas exchange is a function of thenumber of projections, the height of the projections, and the width ofthe device. In this illustration, to demonstrate the versatile range ofdesign options, the first wall of the uppermost culture compartment hasnot been held in position by culture compartment support 42. That ispossible if the culture compartment comes to a horizontal state iffilled with a fluid, or a pressurized fluid, or if it is comprised of astiff material. Also, second wall 120 of the culture compartment neednot make contact with the culture compartment support if it is comprisedof a stiff enough material to retain its shape when medium resideswithin it.

The culture compartments themselves can be structured to perform therole of allowing ambient gas to communicate with the neighboring culturecompartment while maintaining desired geometry. Wilson et al. 5,693,537describe how a wall with projections can be used to provide support foran adjacent wall of the culture compartment. FIG. 10 shows across-sectional view that provides one example of how the desired shapecan be maintained. In this example, first wall 110 of gas permeablemulti-shelf device 7 is formed of a rigid material and the gas permeablesecond wall 120 is comprised of a flexible material such as dimethylsilicone. Wall projections 150 emanate from the surface of second wall120 in order to maintain gas space 50. Wilson et al. 5,714,384 show howthe projections can be used to increase the surface area for gastransfer. It will be understood by skilled artisans that the projectionscould emanate from the surface of the second wall to make contact withthe first wall of the neighboring culture compartment, or from the upperand second walls. Alternatively, projections emanating from the outsidesurfaces of the walls of the culture compartments could interlock witheach other, or with the culture compartments supports. As anotherapproach, all walls can be flexible, and they can take the desired shapewhen medium fills the culture compartments.

The upper and lower walls, and/or upper and lower culture surface, of agiven culture compartment should not make contact with each other. Forexample, when one culture surface is tissue culture treated and contactwith the opposing wall can potentially affect the tissue culture treatedsurface, an internal spacer can be placed within the culturecompartments to ensure contact is prevented. The internal spacer can beany biocompatible material and should be configured to allow medium andfluid to easily move into and out of the culture compartment.

The internal spacer need not be a separate part, as maintaining thedesired space between any walls and/or culture surfaces, can be achievedby projections emanating from the upper and/or lower walls, and/or upperand/or lower culture surfaces. FIG. 11 shows an illustrative example inwhich internal spacer 160 of gas permeable multi-shelf device 8 is aboss that emanates from culture surface 130 or wall 110. Those skilledin the art will recognize that the internal spacer can be constructed ina wide variety of ways, provided that those ways do not prevent mediumfrom entering or exiting the culture compartments.

The ability to microscopically observe cells in culture can be impededwhen the culture compartments are stacked vertically, as the light isdiminished. Thus, offsetting a culture compartment from the stack, asdescribed in co-pending Wilson et al. '814 can be useful in allowing theuse of an inverted microscope. Another option is to make the gas spacecapable of receiving light so that inverted microscopic observation ispossible. To do so, the distance between the culture compartments shouldbe great enough to allow a light source to illuminate the contents ofthe lowest culture compartment. The intensity of the light will dependupon the materials of the culture compartment and the height of themedium. Optically clear materials are preferred.

FIG. 12 illustrates an embodiment that connects culture compartments 20in series.

Liquid delivered into gas permeable multi-shelved device 9 by way ofaccess port 70 displaces gas by way of another access port 70 and comesto reside in any desired number of culture compartments 20. A gas space50, in communication with ambient gas, is present between culturecompartments 20 and adjacent to gas permeable materials. In thisillustration, gas space 50 is present in openings through the entirebody of gas permeable multi-shelf device 9.

In some cases there may be a desire to access each culture compartmentindividually, even though they are integral to a common device, such aswhen each culture compartment contains different cell types, or adifferent medium composition for a common cell type. Doing so can beaccomplished by a variety of configurations. Preferably, the access toeach culture compartment is structured so that it can be accomplished bystandard liquid handling approaches such as pipetting or pouring, oraseptic or closed system approaches like septums or sterile tubingconnections. One option is shown in FIG. 13 . Culture compartments 20 ofgas permeable multi-shelf device 10 can be individually accessed by wayof access ports 70, in this illustration shown in a septum format. Oneor more access ports 70 can be connected to each culture compartment 20.In this illustration, gas space 50 is structured so that a user canallow it to be in communication with ambient gas, or prevent itscommunication with ambient gas. Gas space 50 is enclosed by gas spacehousing 51. Gas space access openings 55 in gas space housing 51 allowgas space 50 to communicate with the ambient environment. Gas spaceaccess openings 55 can be structured to be open and closed as desired.The ability to selectively terminate, restrict, or open gas movementbetween gas space 50 and ambient gas can be useful. This feature can bepresent in any embodiment. For example, when the gas permeablemulti-shelf device is temporarily removed from a CO2 environment,closing or restricting gas space access openings 55 can prevent or delaya shift in pH. As another example, cells of a given cell line can beplaced in each compartment, the gas space in communication with a givencompartment can be primed with a predetermined oxygen concentration, thegas space can be closed, and the effect of each oxygen concentrations oncell growth and/or function can be studied. Those skilled in the artwill recognize that a wide variety of methods for opening and closinggas space access openings 55 are available, including luer openings andplugs, ports and caps, and the like.

FIG. 14 shows another embodiment of the gas permeable multi-shelf deviceconfigured to limit the rate of pH change when the device is removedfrom a standard tissue culture incubator for liquid handling in a flowhood. Gas space housing 51 encloses gas space 50 of gas permeablemulti-shelf device 11. Gas space 50 can be isolated from the ambient gasby closing gas access cover 53 of opening 55 prior to removal from theincubator, thereby trapping the desired level of CO2 in gas space 50.Preferably, the volume of gas in gas space 50 is enough to support theoxygen demand of the culture during the time period that gas accesscover 55 is closed. Therefore, the number of cells or tissue present inthe device, in addition to the oxygen demand, is a consideration foroptimal volume determination.

FIG. 15 shows another embodiment of the gas permeable multi-shelf deviceconfigured to limit the rate of pH change when the device is removedfrom a standard tissue culture incubator for liquid handling in a flowhood. In this case, gas space 50 is open to ambient gas along one sideof the gas permeable multi-shelf device. Gas exchange control rim 57extends from that side of gas permeable multi-shelf device 12, in thisillustration as a feature of gas space housing 51. When gas permeablemulti-shelf device 12 is oriented such that gas exchange control rim 57is flush to a flat surface, such as the floor of a laminar flow hood,gas exchange between gas space 50 and ambient gas is terminated orsubstantially restricted, thereby reducing the rate of pH shift. Anothersimple method of minimizing the rate of pH shift when the gas permeablemulti-shelf device does not have features to control the shift of pH andit is removed from the incubator is to place it in an enclosure such asa box with a lid. The lid need not be gas tight to provide an advantage.So long as the cross-sectional opening of gas access between the lid andthe box is less than the cross-sectional opening between the gas spaceand the ambient gas, a restriction in the rate of gas exchange, and adelay in pH shift will occur. The gas volume in the box when the gaspermeable multi-shelf device resides within it should be minimized.

Furthermore, the box can be preconditioned to contain the gascomposition of the incubator prior to placing the gas permeablemulti-shelf device within it.

The novel gas permeable multi-shelf device allows protocols not possiblein traditional multiple shelf flasks. For example, cells can be expandedfrom one shelf to others. A cycle of inoculating, expanding, andharvesting an adherent cell population that is not adversely affected byresidual trypsin provides one example of how the closed system processcan function. FIG. 16A shows a cross-sectional view of gas permeablemulti-shelf device 13 with medium 80 and cells residing in the lowerculture compartment 20. To expand the adherent cells to the number ofculture compartments shown in FIG. 16B, a straightforward sequence ofevents can take place. First, medium 80 is removed. PBS is thenintroduced into the lower culture compartment to flush residual medium.Subsequently PBS is removed. Trypsin, or any other detachment material,is then introduced into the lower culture compartment to release cellsfrom the attachment surface. Cells can then be redistributed to upperculture compartments by the addition of medium 80, which dilutes trypsinto a level that does not affect cell attachment. If cells are affectedby any residual trypsin, the cells can be removed and it can becentrifuged out using conventional means. Then the cells can bereintroduced in an appropriate volume of medium such that they come toreside in the desired number of culture compartments. With suspensioncells, the expansion to additional culture compartments can be as easyas simply adding an appropriate volume of medium. In this manner, theuse of ancillary devices to create inoculum is minimized relative totraditional multiple shelf flasks, greatly simplifying the cultureprocess. Since the gas permeable multi-shelf device can be configuredfor closed system access, the probability of contamination is alsoreduced.

The ability for cells to reside on sidewall surfaces also createsadvantages that include the ability to expand cells from a surface areaof one size to surfaces of increased size. For example, when byorienting the gas permeable multi-shelf device in the position shown inFIG. 5C, the culture can be initiated using a small quantity ofinoculum, which will reside in proximity of walls 126. Then, when thepopulation of the culture has expanded, more surface area can be madeavailable by reorienting the device to the position of FIG. 5B. Ifneeded, cells can be trypsinized from walls 126 prior to reorienting forincreased surface area. If more expansion is needed, the device can thenbe reoriented again to the position of FIG. 5A Subsequently, furtherexpansion is possible by the methods described in the previousparagraph.

Any material normally associated with cell culture devices or medicaldevices can be used throughout the gas permeable multi-shelf device.Preferably, material that is selected meets the USP VI and/or ISO 10993standard for compatibility. Also, optical transparency is desirable asit allows visual detection of contamination and pH. When creatingsurfaces that are to be observed via inverted microscope, a SPE 2surface or better is preferred.

The gas permeable material used to allow gas transfer into and out ofthe gas permeable multi-shelved device can be comprised of any membrane,film, material, or combination of materials used, or previouslydescribed for use, in gas permeable cell culture devices, such assilicone, flouroethylenepolypropylene, polyolefin, polystyrene film, andethylene vinyl acetate copolymer. Many sources for learning about gaspermeable materials and their use in cell culture are available forguidance, including but not limited to U.S. Pat. Nos. 5,693,537,6,455,310, 6,297,046, International Publication Number WO 01/92462, andco-pending U.S. patent application Ser. No. 10/961,814. An additionalsource of information can be found in the Plastic Design Library,William Andrew Publishing, “Permeability and Other Film Properties ofPlastics and Elastomers”, 1995. The use of the word silicone throughoutthis specification includes the formulations described in U.S. Pat. No.6,045,877.

As described in Wilson et al. 5,693,537, the gas permeable material maybe a liquid permeable material. Those materials include membranes thatare hydrophilic throughout the cross-section, such as those comprised ofcellulose, cellulose acetate, and regenerated cellulose. However, inexperiments that evaluated the use of such material, it was discoveredthat measures for the prevention of contamination, not anticipated inWilson et al. '537 are preferred. Care should be taken to ensure thatthe material selected has a low enough liquid permeability to retain adesired volume of medium within the culture compartments. Moreover,liquid loss can increase osmolarity to a detrimental level. Preferably,a material that is selected will have the ability to retain over about90% of the medium volume in the culture compartment for the intervalbetween feeding, at the given static pressure of the medium. Duringfeeding, osmolarity can be restored. In the case of two-day feedingintervals, liquid loss due to static pressure should thereforepreferably be limited to a ratio less than about 5% per day of mediumvolume within the culture device. For example, it has been discoveredthat 10,000 molecular weight cutoff, 80M CUPRAPHAN® membrane is anacceptable material at medium volumes of at least 10.16 ml of medium percm² of membrane. The material is also thin, and capable of providingadequate gas transfer. In an experiment conducted in CELLine CL1000product fabricated by Wilson Wolf Manufacturing with the lower gaspermeable material composed of 80M CUPRAPHAN®, the ability to culture atleast 400×106 murine hybridoma cells upon was demonstrated. Other thanusing 80M CUPRAPHAN® as the lower gas permeable membrane, all otheraspects of the device were the same as the commercially availableproduct, which integrates a non-liquid permeable, gas permeablemembrane. In this experiment, the surface density was at least 4×106cells/cm² of gas permeable membrane. However, although no contaminationwas detected within the culture compartment, the outside of the membranebecame contaminated. Thus, constructing the gas permeable multi-shelfdevice with gas permeable, liquid permeable material should preferablyrestrict access to the gas space by the use of gas space access openingsto the gas space that are covered with a sterile filter. Any gaspermeable filtration material typically used to prevent contaminationsuch as microporous membranes can be used. To best preventcontamination, pore size can range from 0.45 μm down, and is preferablyat 0.2 μm. However, the use of gas permeable liquid impermeable materialis not limited to just the gas permeable multi-shelf device embodiments.Other gas permeable configurations, including those as simple, forexample as the OPTICELL product (partially described in U.S. Pat. No.6,821,772) could integrate at least one gas permeable, liquid permeablemembrane such as CUPRAPHAN®. As another example, the Slide-A-LyzerDialysis Cassettes (U.S. Pat. No. 5,503,741), normally not associatedwith cell culture, could be used as a culture device with a preferredconfiguration that included a gas space in contact with either, or both,of the dialysis membranes, and by the use of gas space access openingsto the gas space that are covered with a sterile filter.

When configuring the gas permeable multi-shelf device such that it canbe oriented in a first position in which suspension cells are cultured,or oriented in an alternative position in which adherent cells arecultured, a preferred configuration of construction of the gas permeablemulti-shelf device should be such that one culture surface of the cellcompartments is hydrophobic and a different surface is hydrophilic. Anexample can be illustrated by any of the cross-sectional drawing. Forinstance, referring to FIG. 4 , first wall 110 could be gas permeableand its inner surface could be tissue culture treated to create oneculture surface, while the inner surface of second wall 120 could behydrophobic to create another culture surface. Thus, to culture adherentcells, the gas permeable multi-shelf device would be operated with firstwall 110 residing below second wall 120. Thus, to culture suspensioncells, the gas permeable multi-shelf device would be operated withsecond wall 120 residing below first wall 110. Co-culture could beconducted when adherent cells attached to first wall 110, and then thedevice is reoriented to allow suspension cells to reside upon secondwall 120. A useful material to culture suspension cells upon issilicone, and a typical material to culture adherent cells upon tissuetreated polystyrene. Therefore, in this example, a preferred embodimentwould be where second wall 120 is comprised of silicone, and the culturesurface of first wall 110 is comprised of tissue culture treatedpolystyrene. However, it was discovered that if silicone is used, it canmigrate during gamma irradiation ore-beam sterilization and coat tissuetreated surface within the culture compartments. This renders the tissuetreated surfaces suboptimal for adherent cells.

Thus, popular methods of sterilization are not practical when the mostuseful materials are present. Other methods of sterilization areproblematic. For example, ETO will be retained in the silicone, andwithout a very extensive flush of the toxins, an unhealthy environmentfor cells will exist. Chemical means of sterilization are also require aflush. Attempts to correct the problem through the addition of colorantto the silicone, and/or variations in cure temperature and time, and/orpre-exposing the silicone to gamma irradiation at high doses, and/orchanging the distance from the silicone to the polystyrene did noteliminate the problem. However, it was discovered that plasma chargingthe silicone prior to submitting the device to gamma irradiation showedthe ability to minimize or eliminate the migration of silicone onto thepolystyrene surfaces.

Therefore, a preferred process of using silicone in the presence oftissue culture treated surfaces, preferably polystyrene, is to ensurethat the silicone is plasma charged prior to gamma irradiation. Thisapproach to the formation of a cell culture device is not limited to thegas permeable multi-shelf device. This approach allows any gas permeableculture device to integrate plasma charged silicone in the presence oftissue treated surfaces, with the benefit of preventing migration ofsilicone during traditional sterilization methods such as gammairradiation or e-beam. For example, the devices described by Wilson etal. '814 or in U.S. Pat. No. 6,821,772 would benefit by the use ofplasma charged silicone in the presence of treated surfaces. Forexample, the commercially available OPTICELL™ product could integrateone gas permeable tissue culture treated polystyrene surface and anopposing gas permeable surface comprised of plasma charged silicone. Inthis manner, when sterilized by standard methods, suspension cells couldbe cultured upon the surface comprised of silicone and/or adherent cellscould be cultured upon the surface comprised of polystyrene. The productcould integrate traditional distances between membranes, as currently isthe case, or increased distances as described in Wilson et al. '814.

FIG. 17A shows a view of a culture compartment molded of silicone thatminimizes the number of joints for leakage. The use of dimethyl siliconeis advantageous, as it can be molded in complex geometric patterns andit is beneficial in many suspension cell culture applications. Culturecompartment 20′ includes flange 21. In this configuration, it ispossible to eliminate joints at the intersection of the walls. Thus, itis an integral culture compartment that allows modular device design.Flange 21 can be glued to another silicone surface, or secured to anysurface, such that a series of culture compartments are ready forattachment to manifold walls. Alternatively, a series of culturecompartments can be prepared for assembly to a manifold wall by placinga rigid plate in front of the flanges, a rigid plate behind the flanges,attaching the rigid plates together, and then mating the subassembly tothe manifold wall. Any alternative culture surfaces can be placed intothe culture compartments prior to assembly. The integral culturecompartment need not be limited to just one culture compartment. Morethan one culture compartment can be molded as an integral piece to formintegral culture compartments that can also allow modular assembly.Although thickness in the areas where gas transmission is desired ispreferably less than or equal to about 0.022 inches, and more preferably0.010 inches, and most preferably less than about 0.008 inches forhighly demanding cultures, greater thickness can also be useful whencells do not exhibit high oxygen demand. Thicker cross-sections ofsilicone can be helpful in fabrication. In general, the thicker thesilicone, the easier it is to fabricate. However, the ability tofabricate an integral culture compartment of dimethyl silicone, withupper and lower walls at about 0.007 inches thick, was established. Theability to fabricate an integral culture compartment of dimethylsilicone, with sidewalls at about 0.004 inches thick was alsoestablished. Although depicted with more than one flange, the integralculture compartments need not have more than one flange. Thus, the canbe dead-ended when only one manifold is desired.

In a preferred approach, all culture compartments are molded as anintegral piece with a common flange that can be secured to a manifoldwall. FIG. 17B shows integral culture compartments 20″, which are aseries of molded gas permeable material compartments, preferablydimethyl silicone, including a flange 152. A rigid piece of plasticacting as a flange support, such as polycarbonate, can be moldeddirectly (i.e. over-molded) onto the flange to act as a backing forsubsequent further assembly. In this depiction, a flange support 153 isover-molded onto each flange 152. As shown in the exploded assembly viewof FIG. 17C, flange support 153 mates to manifold wall 62 in a liquidtight manner to form a manifold on each end of the culture compartments.When evaluating the capacity to bond dimethly silicone to a rigidplastic, a liquid tight bond was obtained between dimethyl silicone andbiocompatible polycarbonate. The polycarbonate material was placed in amold and silicone was mated directly to the polycarbonate in a liquidinjection molding process. Culture compartment supports can be added,such as any the range of configurations described herein and/or shown inFIG. 10 and FIG. 11 . If created as described in FIG. 11 , theprojections can be ribs extended across the entire culture compartment.In the illustration of FIG. 17C, a series of culture compartmentsupports 42′ are present. Even without need of further modification byintroduction of a culture surface, this is a useful configuration for awide variety of suspension cells. However, the addition of culturesurfaces remains optional. When the intended application may include theuse of adherent cells, it is preferred that the inner surface of thesilicone by covered with a typical adherent surface appropriate for thetype of cells being cultured. For a wide variety of adherent cells,inserting a layer of tissue treated polystyrene as a culture surface canbe useful. That allows a wider variety of culture protocols. Forexample, to culture both adherent and suspension cells, the device wouldbe oriented to allow adherent cells to gravitate to the tissue treatedculture surface, and post attachment, rotated one-hundred-eighty degreesto allow suspension cells to reside upon the opposing silicone wall.

If the configurations of FIG. 17 are created with the intent ofincluding a tissue treated culture surface other than silicone, plasmacharging the silicone to prevent migration during e-beam or gammasterilization can be avoided by covering all the inner silicone surfaceswith a different culture surface. For example, thin tissue treatedpolystyrene can be inserted in a manner that covers all of the silicone.When manifold wall is not a material that migrates, the treatedpolystyrene surfaces will remain suitable for adherent culture. Althoughthe culture surface, in this example polystyrene, provides a surface forcells to attach to, it impedes gas transfer into the cell culturecompartment relative to what it would be if it were just silicone. Thus,the culture surface (or surfaces) that cells are intended to reside uponshould be thin so that gas transfer can still be adequate. Preferably,the thickness in the area of the polystyrene that cells reside upon isabout 0.003 inches or less. This approach reduces the number of jointsand the potential for leakage, while providing culture surfaces that aresuitable for any given application.

FIG. 18C FIG. 18D, and FIG. 18E illustrate the advantages of anotherembodiment that allows culture compartments to be partially filled withmedium, and/or minimizes the volume of trypsin, PBS, or any other liquidthat is involved in removing cells from the cell compartments. In thisembodiment, the advantages of the present invention are integrateddirectly into the format of the traditional multiple shelf cultureflasks such as the NUNC Cell Factory, NUNC Triple Flask, and CORNING®CELLSTACK®. FIG. 18A is a drawing provided on the website of NUNC,depicting the operation of their Cell Factory for Active Gassing, whichis intended to overcome the problems of non-uniform gas composition. Thegassing system complicates the process, requiring tubing connections,sterile filters, a separate gas supply, and an option for humidificationsince a continuous flow of dry gas mix can quickly evaporate medium andincrease osmolarity to levels that affect cell growth and function. FIG.18B is a magnified view of the NUNC approach of FIG. 18A, with arrowsindicating forced gas flow and walls 224 and 222 identified forreference. The gas permeable multi-shelf flask can address the problemof non-uniform gas composition without the need for the ancillaryequipment or forced gas flow required for traditional multiple shelfflasks. An advantage of the embodiments described in FIG. 18C, FIG. 18D,and FIG. 18E is that they can integrate the features present intraditional multiple shelf flasks, such as those commercially availableand such as those described in U.S. Pat. Nos. 5,310,676, 6,569,675, andUK Patent Specification GB 1539263A, including any or all features thatallow medium to be distributed equally to each shelf FIG. 18C shows anembodiment of gas permeable multi-shelf flask 200 in which uniformculture conditions are attained without need of ancillary equipment orforced gas flow required for traditional multiple shelf flasks. At leasta portion of wall 224 and/or wall 222 is gas permeable, allowing gascommunication between gas space 250 and ambient gas. Traditional culturecompartments can reside in the device, preferably with shelf 220structured of polystyrene. Culture compartment walls 220 are structuredat a traditional height to retain medium and cells such that cells 90receive oxygen as a result of gas transfer at the interface betweenmedium 80 and gas space 250. Manifold 260 can be structured as in thatof the traditional flask. Dimethyl silicone is a good choice for gaspermeable material because is can be liquid injection molded as ahousing, or it can be over-molded onto rigid walls such aspolycarbonate. However, as described previously, the silicone should beplasma charged in the event that gamma irradiation is anticipated.Preferably, the gas permeable area of the walls is distributed in anequal geometric relationship to each culture compartment, therebyallowing each culture compartment equivalent access to gas transfer.Stated differently, each culture compartment should reside an equaldistance from gas permeable material. In the area of gas permeablematerial, protection from damage and structural support can be providedso long as the component providing those attributes allows gas access tothe gas space. Thus, optional sidewall support 240 performs thatfunction. It is comprised of gas access openings 242 and/or projections241. Preferably, sidewall support is a rigid, clear material.

FIG. 18D shows another approach that eliminates the need for forcedgassing as an approach to providing uniform culture conditions intraditional multiple shelved devices. This configuration integrates gaspermeable materials within the body of the device. Gas space 251 is anopening through at least a portion of the body of gas permeablemulti-shelf device 201. Thus, gas space 251 is bounded by upper gasspace wall 210 and lower gas space wall 215. It can be an openingthroughout the entire body, similar to the illustration shown in FIG. 8Aand FIG. 8B. It need not pass entirely through the device, and canterminate within the device. Any of its walls can be gas permeable.However, upper gas space wall 210 need not be gas permeable to overcomethe non-uniform culture conditions in the traditional multi-shelf flask.That can be achieved when any of the other walls of gas space 251 arecomprised of gas permeable material. For example, upper gas space wall210 can be polystyrene, and of a thickness that does not provideadequate gas transfer so long as at least another wall(s) of gas space251 is comprised of material that does provide adequate gas transfer.Gas transfer at uniform locations within the body of the device affordstraditional cell culture using a gas-liquid interface for gas exchangewith a more uniform gas environment than that of traditional multipleshelf flasks. The gas permeable multi-shelf device need not only beoperated in a manner of the traditional multiple shelf flask. Gastransfer can take place independent of a gas-liquid interface if uppergas space wall 210 is comprised of gas permeable material. Upper gasspace wall 210 can mate with sidewall 225 to form a cell culturecompartment. Then, for example, if there is a desire to increase mediumheight to minimize feeding frequency or delay a shift in osmolarity dueto evaporation, medium can be added to any height desired so long as theheight of culture compartment sidewalls 225 is increased accordingly asdescribed in Wilson et al. '314.

FIG. 18E shows an illustration of an adaptation of a traditionalmultiple shelf flask to create a gas permeable multi-shelf flask that isconfigured with gas permeable wall for cells to reside upon when gastransfer through the sidewall of the device occurs. This provides moreoptions for culture methods and addresses the issue of non-uniformculture conditions when compared to the traditional multiple shelfflask. Gas permeable multi-shelf flask 202 is configured with at least aportion of its walls, such as sidewall 224 and 222, comprised of gaspermeable material. If needed, areas that are gas permeable should besupported as described in FIG. 18C. Referring again to FIG. 18E, culturecompartment bottom 245 is comprised of gas permeable material andpreferably is supported by culture compartment support 243, which mayinclude projections 241 and/or gas access openings 242. Culturecompartment walls 225 can be at whatever height is needed to allowmedium 80 to reside at the desired height. Manifold 260 can bestructured as that of the traditional flask.

EXAMPLES

Example I and Example 2 assessed alternate geometry of the culturecompartment support in order to demonstrate quantitatively how the gaspermeable multi-shelf flask has the capacity to resolve the traditionalflasks excessive use of shipping, sterilization, storage, incubator, anddisposal space while simultaneously minimizing the potential fornon-uniform culture conditions to exist.

Example 3 describes how plasma charging silicone prior to gammairradiation can limit or prevent its migration onto tissue culturetreated polystyrene surfaces, thereby allowing silicone and tissueculture treated plastics to co-exist in the same culture compartmentwithout need to deviate from standard sterilization processes.

Example I

Culture compartment support structures for cultures with very highoxygen demand.

The physical structure of a culture compartment support that would allowan improvement in islet culture, known to be one of the highest types ofcultures for oxygen demand, was demonstrated by constructing a testfixture that had its lower wall comprised of a molded dimethyl siliconesheet with an average thickness measured at about 0.0072 inches thickand a surface area of 98 cm². Gas transmission of the dimethyl siliconerubber was determined by MOCON (Minneapolis, MN) using their Oxtran 2/21Instrument in accordance with ASTM-1927 to be about 14,300 m102/100in2/24 hours at 37° C. The culture compartment that supported thedimethyl silicone consisted of a 0.048 cm thick, 46% open, mesh indirect contact with the silicone. The open mesh was comprised of aseries of polypropylene strands, each with a diameter of between0.018-0.020 inches thick, arranged vertically and horizontally such that16 strands were present per inch of horizontal distance and per inch ofvertical distance. The mesh was held in place by a molded polycarbonateplastic sheet of a thickness of 0.19 cm, with uniformly distributedprojections that elevated the mesh above the sheet so that a gas spaceresided below the membrane. Each projection was a uniformly shaped “Y”,while each leg of the “Y” oriented 120 degrees apart. The length of eachleg was 0.45 cm and the width was 0.127 cm. Thus, the surface area ofeach projection available to support the mesh was about 0.175 cm².

About 1.1 projections resided per cm². Thus, the cumulative surface areaof the projections available to support the mesh was about 18.87 cm².The height of each projection was 0.127 cm from the plastic sheet. A gasspace resided between the bottom of the silicone and the top of theplastic sheet. The cumulative volume of gas displaced by the projectionswas 2.4 cm³. The cumulative volume of gas displaced by the mesh was 2.54cm³. Therefore, the gas residing underneath the silicone membrane andabove the plastic sheet was about 17.2 ml. The ratio of the gas residingunderneath the silicone membrane and above the plastic sheet to gaspermeable membrane surface area was 17.6%. The plastic sheet includedthrough holes, acting as gas access openings, the cross-section of eachbeing oriented perpendicular to the plane of the plastic sheet, for thepurpose of allowing ambient gas to communicate with the gas space bypassive diffusion. Five uniformly spaced through holes resided below the98 cm² surface area of the dimethyl silicone, each hole having across-sectional area of 0.29 cm² and a length of 0.075 in, created acumulative cross-sectional area of 1.45 cm². Thus, the ratio of thecross-sectional area of the through holes to the cross-sectional area ofthe silicone membrane was about 1.45 cm^(2/98) cm², or about 1.48%. Theratio of the cross-sectional area of the through holes to the volume ofgas residing between the silicone membrane and the upper surface of theplastic sheet was thus 1.45 cm2/l 7.2 ml, or about 8.4%. Feet elevatedthe bottom of the plastic sheet 0.51 cm. Thus, the total height of theculture compartment support residing below the silicone membrane was0.87 cm.

The following definitions and abbreviations are useful for understandingislet assessment:

-   -   Flask Control . . . A device that relied on a gas-liquid        interface for oxygenation, seeded at a maximum of 200 IE/cm²        with an IE to medium ratio of 1000 IE/ml to yield a maximum        medium depth of 0.2 cm. This control is used to compare the GP        Device to standard islet culture methods in flasks.    -   GP device . . . Test device configured with a bottom of        gas-permeable dimethyl silicone comprising a surface area of 98        cm² and supported by the structure described in Example 1 or        Example 2.    -   IE (Islet Equivalent) . . . A measure of islet volume, equal to        the volume of a 150 μm diameter islet. As the vasculature of a        freshly isolated islet collapses, its volume decreases and its        density increases. So an IE has the same volume but not the same        mass on day O as on day 2.    -   IE by DNA or DNA IE . . . An indirect measure of islet mass,        equal to 11.4 ng DNA    -   IE by Manual Counts . . . IE numbers are traditionally measured        by manual counts which ignore how flat or dense the islets are.        Day O IE by DNA were 63±12% of IE by manual counts in 18 porcine        islet isolations (range 49-93%). Numbers usually converge as        islet volume drops in culture but this is not always the case as        manual counts are prone to errors. Unless otherwise noted, IE        refers to an IE measured traditionally by manual counts.    -   Islet Fractional Viability . . . The fraction of islet mass that        is viable.    -   Islet Surface Density . . . The volume of islets cultured upon a        given surface area, expressed as IE/cm2. A confluent square        array of 150 μm diameter islets has 4444 IE/cm2.    -   Medium Dilution . . . The ratio of medium volume to number of        islets residing in a device, expressed as μ1/IE.    -   Non-GP device . . . A control device configured with identical        geometry as the GP Device, but without a gas-permeable membrane        (used as an experimental control with identical culture        conditions as the GP Device to quantify the benefit of the gas        permeable membrane feature).    -   Porcine Isolation . . . The process of obtaining islets from the        pancreas of pigs using the Ricardi Method.    -   OCR . . . Oxygen Consumption Rate, expressed as nmol/min. A        measure of viable islet mass.    -   OCR/DNA . . . OCR per DNA content, expressed as nmol/min·mg DNA.    -   p Value . . . Reported p values are for the two-tailed paired        Student's t-Test.    -   Recovery . . . The fraction of an islet attribute (e.g., DNA,        IE, OCR) remaining present at a later time.

An initial assessment was conducted using porcine islets to determinewhat the ratio of medium volume to IE would be needed. Porcine isletswere cultured at 37° C. for 2 days in small GP devices with a dimethylsilicone surface area of 18 cm2, at 200 IE/cm2 and medium dilutions at 1μl/IE and 4 μI/IE showed no statistical difference in islet viability asassessed by OCR/DNA For 5 porcine isolations, the OCR/DNA at 4 μ1/IEranged from 97.5% to 102.4% of that at I μI/IE, with the combinedaverage at 10 I %. Based on this finding, a medium dilution ratio of Iμ1/IE was used for the bulk of the evaluations described in Example Iand Example 2.

Islets from 10 porcine isolations were used in a series of experiments,with the primary objective of determining if surface density beyondconventional methods, ranging from about 1000 IE/cm2 to 2551 IE/cm2 bymanual counts (490 IE/cm2 to 2551 IE/cm2 by DNA counts) in the GPdevices could be achieved without loss in fractional viability relativeto flask controls (i.e. gas-liquid interface) at conventional surfacedensity less than about 200 IE/cm2 by manual counts. Non-GP devicescontrols were present with the hypothesis that a compartment supportstructure that only rendered the surface that cells resided uponhorizontal, and not providing gas delivery, would render poor isletviability. In question was the ability of the culture compartmentsupport, structured as described above, to allow adequate oxygendelivery to the islets while managing to maintain islets in a uniformdistribution absent the loss of health from aggregation. The GP deviceswere structured such that islets were uniformly distributed across the98 cm2 surface of dimethyl silicone. Average islet surface density in GPdevices was 1526 IE/cm2 by manual counts. Based upon the ratio offractional viability of GP devices to that of representative flaskcontrols, GP devices showed equal viability with a standard deviation of9.4% and a p value of 0.9987. Thus, the ability for the culturecompartment support to allow passive gas transfer into the culturecompartment at a rate that allowed at least an average 7-fold increasein surface density relative to traditional methods without loss of isletviability as determined by OCR/DNA was demonstrated. This demonstratesthat a culture compartment support can be structured to allow ambientgas to be present on the opposite side of a culture compartment supportrelative to the gas permeable surface in proximity of the culturecompartment support, passively move along the culture compartmentsupport, then perpendicular to the surface upon which cells reside, andthen passively circulate below the gas permeable surface upon whichcells reside while providing enough oxygen transfer to support islets atbeyond seven times that allowed in traditional culture devices.

Example 2

A different physical structure of a culture compartment support thanthat of Example 1 was examined in another islet culture application. Inthis example, test fixtures included virtually identical gas permeablematerial as that of Example 1. The culture compartment that supportedthe dimethyl silicone consisted of an open mesh in direct contact withthe silicone, and a machined polycarbonate plastic sheet supported themesh in a generally horizontal position.

Unlike the culture compartment support of Example 1, the mesh resideddirectly upon the upper surface of the plastic sheet. The mesh geometryand material composition was identical to that of Example 1. For eachcm2 of silicone membrane surface area, the volume of gas between thelower surface of the silicone and the upper surface of the plasticbottom, after displacement by the mesh, was 0.022 ml. Stateddifferently, the ratio of gas volume between the plastic sheet and thegas permeable membrane to the surface area of the gas permeable membranewas 2.2%. In order to allow ambient gas to communicate with the gasspace by passive diffusion, through holes, acting as gas accessopenings, were present in the plastic bottom, the cross-section of eachbeing oriented perpendicular to the plane of the mesh. Each through holehad a diameter of 0.125 inches. The through holes where uniformly spacedin a grid pattern below the dimethyl silicone, such that the distancebetween the center of each hole was 0.375 inches. Each through hole hada length of 0.13 inches. The ratio of the cross-sectional area of thethrough holes to the cross-sectional area of the silicone membrane wasabout 16% of the membrane surface area. The ratio of the cross-sectionalarea of the through holes to the gas volume between the plastic sheetand the gas permeable membrane was 273%. Since the mesh had a height ofabout 0.019 inches, the cumulative distance between the dimethylsilicone and the gas residing under the plastic bottom was about 0.15inches. Eight uniformly distributed feet elevated portions of theperimeter of the plastic bottom 0.41 cm from the surface of the shelfupon which it resided. The perimeter of the bottom was 23.94 cm. Thecross-sectional area between the underside of the plastic bottom and thesurface upon which it resided that was thereby open to movement ofambient gas was 7.59 cm2. Ignoring the feet as a restrictor to gasmovement, the cross-sectional area about the perimeter open to gasmovement to the location of the gas permeable dimethyl silicone was 9.85cm2. Thus, the height of the culture compartment support was about 0.5inches.

Islets from 5 porcine isolations were used in a series of experiments,with the primary objective of determining if surface density beyondconventional, averaging an estimated 1628 IE/cm2 by manual counts (927IE/cm2 by DNA counts) in GP devices could be achieved without loss infractional viability relative to flask controls and non-GP devices. Inquestion was the ability of the culture compartment support, structuredas described above, to allow adequate oxygen delivery to the isletswhile managing to maintain islets in a uniform distribution absent theloss of health from aggregation. If islets were to demonstrate similarviability relative to control as shown in Example 1, the ability tocreate alternative geometry for culture compartment supports would bedemonstrated. A primary difference in geometry is that Example 1utilized projections, whereas Example 2 allowed the mesh to residedirectly upon a flat plastic bottom. To compensate for the lack ofprojections, the geometry of Example 2 had about an 8-fold increase inthe ratio of gas access opening cross-sectional area to gas permeablematerial surface area relative to that of Example 1. Islets weredeposited into the GP devices such that islets were uniformlydistributed across the surface of dimethyl silicone. Based upon theratio of fractional viability of GP devices to that of controlsrepresentative of flasks, GP devices showed identical viability with astandard deviation of 13.8% and a p value of 0.9681. Thus, the abilityfor the alternative geometry of the culture compartment support to allowpassive gas transfer into the culture compartment at a rate that allowedat least an average 8-fold increase in surface density relative totraditional methods without loss of islet viability as determined byOCR/DNA was demonstrated.

The gas permeable test device configuration was also challenged withvery high increases in surface density relative to control, ranging fromabout 7 to 41 times beyond the conventional 200 IE/cm2 surface densitiesof flasks. A total of 20 porcine isolations were evaluated at a surfacedensity averaging roughly 18 times greater than the traditional surfacedensity of flasks. There was a greater degree of variability in thedata, with GP devices exhibiting an average viability of 96.0% of thatof controls representative of flasks, with a standard deviation of 21.9%and a p value of 0.43.

FIG. 19 summarizes the results relative to the non-GP devices and as apercentage of flask control. Clearly, a culture compartment support thatmerely holds the culture compartment horizontal such that islets areuniformly distributed must include the capacity to provide adequate gasexchange, as demonstrated by the loss of islet health with increasingsurface density in the non-GP device. Examples 1 and Example 2 describehow at least two distinct culture compartment supports can allow uniqueadvantages in the efficient use of space when integrated into the gaspermeable multi-shelf device design.

This information is useful in demonstrating the space advantage of thegas permeable multi-shelf device relative to the traditional multipleshelf devices. For example, in the field of islet transplants to curetype 1 diabetes, a goal is to culture up to 800,000 IE as determined bymanual counts. Current flask methods at 200 IE/cm2 surface densitieswould require 4000 cm2 of culture surface area. If using a commerciallyavailable traditional multiple shelf flask, such as the NUNC CellFactory, creating 4000 cm2 of culture surface area would require aboutsix of its 632 cm2 shelves. A NUNC Cell Factor so structured wouldoccupy roughly 416 cubic inches of space and expose islets topotentially non-uniform growth conditions. However, considering theabove examples, a gas permeable multi-shelf device can culture 800,000IE in much less space. For instance, its ability to culture islets at anaverage surface density of about 1526 IE/cm2 to 1628 IE/cm2, allows itto only require a culture surface area of about 500 cm2 to successfullyculture 800,000 IE. If six shelves were used in the gas permeablemulti-shelf device, as required by the NUNC Cell Factory, each shelfwould only need 83 cm2 of surface area. If medium resided directly aboveislets, each culture compartment would be at a height of about 1.6 cm(0.63 in) in order to allow the same feeding frequency as the CellFactory (i.e. 1 μL/IE). The height of the culture compartment supports(i.e. the vertical distance between culture compartments) need notexceed that of the examples. The Examples above demonstrated that eachculture compartment support could be 0.344 in high. Dimensionally, thegas permeable multi-shelf device could be about 5.8 in tall, 3.6 inwide, and 3.6 in long, occupying about 76 in3 of space. That is wellover a 500% reduction in shipping, sterilization, storage, incubator,and disposal space when compared to the 416 in3 of space occupied bytraditional multiple shelf flasks. Furthermore, the non-uniform cultureconditions of the traditional flask are overcome. Note that using aconfiguration such as that shown in FIG. 10 could further reduce thedistance between the culture compartments as demonstrated by the lowfoot distance of the culture compartments of the above examples.

Example 3

Minimizing the migration of silicone during gamma irradiation Testfixture 162 was constructed as shown in the cross-sectional view of FIG.20 . Test sample 165 was fabricated of dimethyl silicone and placed ontothe top of the body of a commercially available polystyrene tissuetreated six-well plate, shown as item 167 (COSTAR® 3516). Thepolystyrene lid 168 was then placed onto six-well plate 167. The abilityto minimize migration of silicone onto inner lid surface 169 and ontotissue culture treated surface 166 by plasma charging test sample 165prior to gamma irradiation was evaluated. Test sample 165 resided at adistance of about 1.78 cm from tissue culture treated surface 166, andless than 2 mm from inner lid surface 169. In one evaluation, testsample 165 was subjected to plasma charging prior to placing it withintest fixture 162 and the presence of the plasma charge was confirmed bya water drop contact angle of ninety-six degrees and a surface energy ofless than thirty dynes. In another evaluation, test sample 165 was notsubjected to plasma charging prior to placing it within test fixture162. In both evaluations, the assemblies were subsequently subjected togamma irradiation. Thereafter, electron spectroscopy for chemicalanalysis (ESCA) was undertaken to quantify the elemental compositions ofvarious surfaces. Tissue culture treated surfaces 166 were assessed forthe presence of silicone, oxygen, and carbon relative to tissue culturetreated surfaces of a control 6-well plate that was gamma irradiatedabsent silicone test sample 165. Inner lid surfaces 169 were assessedfor the presence of silicone. TABLE 1 summarizes the results.

TABLE 1 % % % Test Condition Silicone Oxygen Carbon Tissue culturetreated surface 166 of control 2.309 20.182 77.508 Tissue culturetreated surface 166 in the 2.688 19.549 77.783 presence of plasmacharged silicone test sample 165 Inner lid surface 169 in the presenceof 2.324 6.902 90.049 plasma charged silicone test sample 165 Tissueculture treated surface 166 in the 24.116 50.65 25.243 presence ofnon-plasma charged silicone sample 165 Inner lid surface 169 in thepresence of non- 16.307 59.511 24.183 plasma charged silicone testsample 165

These results show that applying a plasma charge to silicone prior togamma irradiation prevents unwanted silicone migration and surfacestreated for cell culture remain virtually unaltered. The CORNING®six-well plate, gamma irradiated in the absence of silicone (i.e. thecontrol), exhibited the presence of about 20% oxygen on its tissueculture treated surface, as did the CORNING® six-well plate thatintegrated plasma charged silicone. To the contrary, the CORNING®six-well plate that integrated un-plasma charged silicone exhibited agreatly altered oxygen composition, at 51%. Silicone that was not plasmacharged migrated to all surfaces. Silicone that was plasma charged didnot, independent of the proximity of the surface to the silicone.

This opens the door to new configurations of cell culture devices. Ingeneral, a simplified method of fabricating cell culture devices ispossible, including, but not limited to, those described in FIG. 17A andFIG. 17B. A culture device that includes a silicone surface and a tissuetreated surface can be gamma irradiated, with the tissue treated surfacevirtually unaltered post gamma irradiation independent of its distancefrom the silicone, by plasma charging at least the surfaces of thesilicone that are in gas communication with the tissue treated surfaces.Surfaces of silicone that cannot migrate to the tissue treated surfacesneed not be plasma irradiated, such the outside surface of culturecompartments comprised of silicone. Note that surfaces that are onlypartially comprised of silicone should be plasma charged as described,as the silicone portion will migrate during irradiation. In general, asimplified method of fabricating cell culture devices is possible. Forexample, a single compartment device such as a basic flask, or theOPTICELL gas permeable cartridge, can be fabricated by molding asilicone outer housing and inserting a treated polystyrene sheet forcells to reside upon. The unique elongation capabilities of siliconeallow the opening through which the culture surface is added to besmaller than the inserted part, snapping back to a port shape postinsertion. Septum can be present in molded silicone, or other styles ofaccess ports can be present. In the case of the basic flask, the heightof the flask can be substantially reduced as the gas-liquid interfaceapproach to oxygenation is eliminated by the use of gas permeablesilicone. Referring to FIG. 17A, added versatility is obtained whenfirst wall 110 is gas permeable silicone, and culture surface 130 is anadherent surface such as tissue culture treated polystyrene. In thiscase, the device can be oriented first wall 110 down to culturesuspension cells, culture surface 130 down to culture adherent cells, orto culture adherent and suspension cells as previously described. Ifco-culture of adherent cells is desired, an additional culture surfacesuch as very thin, gas permeable polystyrene, can be inserted adjacentto first wall 110. The height of the device can be increased to allowvarious medium volumes to surface area ratios as needed to optimizeculture. Fabrication of the device with pleated sidewalls allows volumeto change as needed by the user. The gas permeable multi-shelf flask canintegrate these benefits also.

Those skilled in the art will appreciate that numerous modifications canbe made thereof without departing from the spirit. Therefore, it is notintended to limit the breadth of the invention to the embodimentsillustrated and described. Rather, the scope of the invention is to bedetermined by the appended claims and their equivalents.

1. A method for culturing cells in a static cell growth apparatuscomprising: adding cells and media to said apparatus; wherein theapparatus comprises only one compartment, said only one compartmentcomprises multiple culture spaces; and said compartment is liquidimpermeable, capable of holding medium, and includes more than one gaspermeable shelf for cells to reside upon; and each shelf has a culturespace between it and an opposing surface; and a manifold connects eachculture space, and the culture spaces reside one above the other whenthe shelves are in a horizontal position; and said apparatus comprisesprojections capable of supporting the outside surface of each shelf andsaid projections are spaced apart to create a space for ambient gas tocontact each shelf; and placing the apparatus in a tissue cultureincubator.
 2. The method of claim 1, wherein the media and cellscompletely fill said compartment.
 3. The method of claim 1, wherein saidopposing surface is gas permeable, liquid impermeable.
 4. The method ofclaim 1, whereby a uniformity of conditions for cellular growth includesa determined media volume per unit surface area.
 5. The method of claim1, wherein said compartment has a substantially rectangular footprintand a substantially uniform height.
 6. A method of culturing cells incell growth apparatus comprising: adding cells and media to said cellgrowth apparatus, wherein said apparatus does not have pumps or otherequipment to perfuse medium or gas; said apparatus is comprised ofmultiple components; said apparatus comprising a liquid impermeablestructure, the inside of which is able to contain cells and medium andthe outside of which is in contact with ambient gas; and more than onesurface on the inside of said liquid impermeable structure being incontact with multiple gas permeable shelves each gas permeable shelfhaving an inside surface and an outside surface; and the inside surfaceof each gas permeable shelf having an opposing surface located adistance away, defining a culture space between each gas permeable shelfand its opposing surface; each culture space having side walls and anopening that allows media and cells to enter and exit the culture space,and the culture spaces are located one above the other when the gaspermeable shelves are in a horizontal position; and a manifold thatconnects the openings of each culture space; projections that areadapted to make contact with the outside surface of each gas permeableshelf when the device is in use, while leaving a portion of the outsidesurface of each gas permeable shelf in contact with ambient gas; andculturing cells in said apparatus in an incubator.
 7. The method ofclaim 6, whereby said liquid impermeable structure is completely filledwith cells and media for optimal cell-nutrient exchange.
 8. The methodof claim 6, wherein said opposing surface is gas permeable, liquidimpermeable.
 9. The method of claim 6, whereby a uniformity ofconditions for cellular growth includes a determined media volume perunit surface area.
 10. A method for culturing cells in a cell growthapparatus comprising: adding cells and media into a cell growthapparatus that is comprised of a single liquid impermeable compartmentthat includes multiple culture spaces, each of which includes one gaspermeable surface for cells to reside upon and an opposing surface; andsaid apparatus includes a manifold that connects each culture space toallow media and cells to go into each culture space; said apparatusincludes a space for gas to make contact with the gas permeable surfaceof each culture space, and placing the apparatus in a tissue cultureincubator wherein the culture spaces reside one above the other andculturing the cells in the presence of ambient incubator gas and withoutperfusing gas or liquid through the apparatus at any time when it is inthe incubator.
 11. The method of claim 1, wherein said media and cellscompletely fill said compartment.
 12. The method of claim 1, whereinsaid opposing surface is gas permeable, liquid impermeable.
 13. Themethod of claim 1, whereby a uniformity of conditions for cellulargrowth includes a determined media volume per unit surface area.
 14. Themethod of claim 1, wherein said compartment has a substantiallyrectangular footprint and a substantially uniform height.